A major challenge for the design of a drug for treatment of any disease is specificity and efficacy. Various drugs available for the treatment of cancer suffer from problems of this nature. The concept of targeting toxic drugs selectively to certain tumors has been a subject of intense research in the last few years (Thorpe (1985) Biol Clin Applications 84:475-512; Moller ed. (1982) Immun. Rev. 62:1-215). Recently both monoclonal and polyclonal antibodies, lectins, lymphokines and hormones which recognize specific determinants on the surface of the tumor cell have been used as carriers to deliver toxic agents into the cell, where the latter can exert their cytotoxic potential (Blattler, et al. (1985) Biochemistry 24:1517-1524; Frankel, et al. (1985) J. Biol, Res, Modif, 4:437-446; Reimann, et al. (1988) J. Clin. Invest. 82:129-138; Schwartz and Vale (1988) Endocrinology 122:1695-1700; Scott, et al. (1987) J Natl. Cancer Inst. 79:1163-1172; Singh, et al. (1989) Biol. Chem. 264:3089-3095; Srinivasan, et al. (1985) FEBS Letters 192:113; Schwartz, et al. (1987) Endocrinology 121:1454-1460). Toxic moieties thus far investigated with these delivery agents include radionuclides (Ghose, et al. (1967) Br, Med, J. 1:90-96), cytotoxic drugs commonly employed in cancer chemotherapy (Thorp and Ross (1982) Immun. Rev, 62:119-157; Deweger, et al. (1982) Immun. Rev. 62:29-45; Arnon and Sela (1982) Immun. Rev. 62:5-27; Pimm, et al. (1982) Cancer Immun. Immunotherap, 12:125-134; Rowland and Axton (1985) Cancer Immun. Immunotherap. 19:1-7) and proteins derived from bacteria and plants such as diptheria or ricin (Jansen, et al. (1982) Immun. Rev. 62:185-216; Raso (1982) Immun. Rev. 62:93-117. Vitetta, et al. (1982) Immun. Rev, 62:159-183; Nelville and Youle (1982) Immun. Rev, 92:47-73; Thorpe, et al. (1981) Eur. J. Biochem. 116:447-454). A specific molecule is designed by replacing the nonspecific B chain with an antibody or a hormone.
Bacterial and plant toxins, such as diphtheria toxin (DT), Pseudomonas aeruginosa toxin A, abrin, ricin, mistletoe, modeccin, and Shigella toxin, are potent cytocidal agents due to their ability to disrupt a critical cellular function. For instance, DT and ricin inhibit cellular protein synthesis by inactivation of elongation factor-2 and inactivation of ribosomal 60s subunits, respectively (Bacterial Toxins and Cell Membranes, Eds. Jelajaszewicz and Wadstrom (1978) Academic Press, p. 291). These toxins are extremely potent because they are enzymes and act catalytically rather than stoichiometrically. The molecules of these toxins are composed of an enzymatically active polypeptide chain or fragment, commonly called "A" chain or fragment, linked to one or more polypeptide chains or fragments, commonly called "B" chains or fragments, that bind the molecule to the cell surface and enable the A chain to reach its site of action, e.g., the cytosol, and carry out its disruptive function. The act of gaining access to the cytosol is called variously "internalization", "intoxication", or "translocation". These protein toxins belong to a class bearing two chains referred to as A and B chains. The B chain has the ability to bind to almost all cells whereas the cytotoxic activity is exhibited by the A chain. It is believed that the A chain must be timely liberated from the B chain-frequently by reduction of a disulfide bond-in order to make the A chain functional. These natural toxins are generally not selective for a given cell or tissue type because their B chains recognize and bind to receptors that are present on a variety of cells.
The availability of a toxin molecule which is not cytotoxic to a variety of cells when administered alone has been limited. Utilizing certain naturally occurring single chain toxin molecules which do not themselves bind to cell surface receptors and, therefore, are not normally internalized by cells, has provided toxic molecules which are relatively non-toxic to most, if not all, cells when administered alone. Such naturally occurring single chain toxins known to date, include, but are not limited to, pokeweed antiviral protein (Ramakrishnan and Houston (1984) Cancer Res. 44:201-208), saponin (Thorpe, et al. (1985) J. Natl, Cancer Inst. 75:151-159), and gelonin (Stirpe, et al (1980) J. Biol. Chem. 255:6947-6953). These proteins are nontoxic to cells in the free form, but can inhibit protein synthesis once they gain entry into the cell. However, the availability of these single chain toxins in substantially pure form is limited due to the fact that they must be purified from plant sources in which they occur in relatively low amounts and the reproducibility of the concentration of the toxin in the plants is dependent upon plant growth conditions and plant harvest conditions.
Gelonin is a single chain polypeptide isolated from seeds of a plant, Gelonium multiforum, having a molecular weight of approximately 28,000-30,000 kd. Gelonin is a basic glycoprotein with an approximate isoelectric point of 8.15 and contains mannose and glucosamine residues (Falasca, et al. (1982) Biochem J, 207:505-509). In contrast to other plant and bacterial toxins, this protein is not toxic to cells by itself, but when delivered to cells through a carrier, it damages the 60s ribosomal subunit. In vivo and in vitro biological data suggest that gelonin is equivalent or superior to other plant toxins. In fact, the results of a comparison of gelonin conjugates in vitro and in vivo with other A chain conjugates indicated that gelonin had similar potency, better selectivity, better tumor localization, and more significant therapeutic effects (Sivan, et al (1987) Cancer Res, 47:3169-3173). However, the availability of a reproducible, readily accessible supply of gelonin from natural sources is limited. In addition, the purification of gelonin from plant sources is difficult and the yield is very low.
Gelonin by itself has been shown to be abortifacient in mice and enhances antibody dependent cell cytotoxicity (Yeung, et al (1988) Internatl. J. Peptide Protein Res, 31:265-268).
Several investigators have utilized gelonin as a cytotoxic agent chemically attached to monoclonal antibodies or to peptide hormone cellular targeting ligands. However, chemical modification of gelonin and cellular targeting moleties can reduce targeting efficiently and cytotoxic potential of gelonin itself. Furthermore, natural sources of gelonin are subject to variability in harvesting and plant growth which can affect gelonin cytotoxic activity. The ability to produce a synthetic gelonin toxin, chemically or utilizing recombinant technology, provides a plentiful, reproducible source of the toxin.